The bispecific antibody (BiAb) is an artificial antibody containing two specific antigen binding sites and can build a bridge between a target cell and a functional molecule (cell) to generate an oriented effector function. The BiAb has a broad application prospect in the biomedicine, especially in immunotherapy of tumors. To kill tumor cells through the BiAb-mediated cytotoxic effect is a hotspot of current application research of immunotherapy, and its principal characteristic lies in that the BiAb can simultaneously bind to a tumor-associated antigen and a target molecule on an immunologic effector cell and directly trigger the specific killing effect of the immunologic effector cell on the tumor cell. However, numerous obstacles, such as difficulty in expression, low yield, difficulty in purification and poor stability generally exist in the research and development process of the bispecific antibody drugs, and therefore, it is very necessary to construct a novel bispecific antibody for overcoming the barriers forementioned and construct a corresponding immunity killing animal model. The present invention provides the construction of the novel bispecific antibody and describes its pharmacological research method and result Immune cell antigens and tumor cell antigens under study and some background arts of related technology development will be introduced below.
1. CD3
The CD3 module consists of four subunits δ, ε, γ and ζ of which the molecular masses are 18.9 kDa, 23.1 kDa, 20.5 kDa and 18.7 kDa respectively and which have 171, 207, 182 and 164 amino acid residues in the length direction respectively. All the subunits constitute six peptide chains which tightly bind to a T cell receptor (TCR) usually to form a TCR-CD3 complex containing eight peptide chains (as shown in structural schematic diagram 1). This complex has the functions of transducing a T cell activation signal and stabilizing a TCR structure. The cytoplasmic domain of CD3 contains immunoreceptor tyrosine-based activation motif (ITAM), and the TCR identifies and binds to an antigen peptide presented by an MHC (major histo-compatibility complex) molecule, resulting in that a tyrosine residue in a conserved sequence of the ITAM of CD3 is phosphorylated by tyrosine protein kinase p561ck in a T cell and then other tyrosine protein kinases (such as ZAP-70) containing SH2 (Scr homology 2) structural domains can be collected. The phosphorylation of ITAM and the binding to ZAP-70 are one of important biochemical reactions in the early stage of the T cell activation signal transduction process. Therefore, the CD3 molecule has the function of transducing the activation signal generated when the TCR recognizes antigens.
2. EpCAM
The EpCAM (CD326) as a specific cell adhesion molecule of an epithelial cell is type I transmembrane glycoprotein. It also refers to some other processes, including cell migration, proliferation, differentiation and the like. EpCAM is one of earliest tumor-associated antigens which are identified by applying a monoclonal antibody technology, is widely expressed onto the epithelial tissue surface in a polymer form, mediates a Ca independent intercellular homotypic adhesion function, and can thus be classified into an adhesion molecule family. EpCAM also has other features of the adhesion molecule family and participates in multiple processes, including interaction and migration of cells and ground substances, cell differentiation, form and cell cycle regulation, signal transduction, metabolization and the like. In the meantime, EpCAM is over-expressed in multiple epithelium-derived tumors, which means that EpCAM is closely related to tumors. In a pathological circumstance, EpCAM is expressed in glandular cancers, including colorectal cancer, gastric adenocarcinoma, breast cancer, ovarian cancer, adenocarcinoma of lung, prostate cancer, pancreatic cancer, hepatocellular carcinoma and retinoblastoma in different degrees. Multiple researches have proved that the expression of EpCAM is related to proliferation, cycle distribution and metastasis of breast cancer and colonic cancer cells (as shown in Table 1). A mono-specific anti-EpCAM monoclonal antibody (MAB), such as a monoclonal antibody 17-1A (glaxowellcome, Centocor) is the first adjuvant therapy approving German EpCAM targeted therapy of colorectal cancer, however, a large amount of clinical medication data displayed that this mono-specific antibody had no remarkable and more beneficial effect compared with chemotherapy. At present, some other EpCAM targeted therapies, including bispecific antibodies, are of a growing trend for cancer therapy, and both the bispecific antibodies MT110 and Catumaxomab are therapeutic bispecific antibody drugs against tumor antigen EpCAM, wherein Catumaxomab has been approved for treating malignant cancer ascites by European Union, and MT110 has been applied in clinical researches. It was obvious that EpCAM had become one of hotshot targets for tumor therapy research at present.
TABLE 1Extensive Distribution of EpCAM in TumorsPositive Rate ofTumorsEpCAMOvarian Cancer88-100%  Gastric Cancer98%Colorectal Cancer99%Pancreatic96%CancerBreast Cancer90%Endometrial91-96%  CancerLung Cancer87%Prostate Cancer98%
3. Technological Development of Bispecific Antibody
The bispecific antibody is an antibody in which two antigen binding sites in one antibody molecule can bind to two different epitopes respectively.
The antibody drug refers to a biomacromolecular drug prepared by an antibody engineering technology taking a cell engineering technology and a genetic engineering technology as main bodies and has the advantages of high specificity, uniform property, capability of realizing directional preparation against specific targets, etc. The monoclonal antibody is mainly applied to the following three aspects in clinical practice: oncotherapy, therapy of immune diseases and anti-infective therapy. Wherein, the oncotherapy is the most extensive field for monoclonal antibody application at present, and products for oncotherapy in monoclonal antibody products that have entered clinical trial and listed in the market account for about 50%. The oncotherapy by monoclonal antibodies is an immunotherapy for killing target cells by stimulating the immune system through binding to specific targets of pathological cells, in order to enhance the effector function of the antibody, and especially the effect of killing tumor cells, and as concerned in multiple methods that have been tried by people to transform antibody molecules, the bispecific body has been one of the development trends for improving the antibody therapy effect and has become the hotspot in the field of antibody engineering researches.
The bispecific antibody for immunotherapy is an artificial antibody containing two kinds of specific antigen binding sites, is capable of building a bridge between the target cell and the functional molecule (cell) and stimulating oriented immunoreaction and has a wide application prospect in immunotherapy of tumors.
4. Preparation of Bispecific Antibody
The bispecific antibody can be obtained by multiple paths, and its preparation methods mainly include a chemical coupling method, a hybrid-hybridoma technique and a genetically engineered antibody preparation method. As concerned in the chemical coupling method, two different monoclonal antibodies are connected together in a chemical coupling manner to prepare a bispecific monoclonal antibody, which is the earliest bispecific monoclonal antibody concept, and the shortcomings of this preparation method are obvious. As concerned in the hybrid-hybridoma technique, the bispecific monoclonal antibody is produced by a cell hybridization method or a ternary hybridoma manner, and these cell hybridomas or ternary hybridomas are obtained through fusion of built hybridomas, or the fusion of the built hybridomas and mouse-derived lymphocytes, could only produce a mouse-derived bispecific antibody, and are thus limited to a great extent in application. With the rapid development of the molecular biological technology, multiple construction modes of humanized bispecific antibodies in genetic engineering have arisen, which are mainly classified into four categories, namely a bispecific micro-antibody, a double-chain antibody, a single-chain bivalent antibody and a multivalent bispecific antibody. At present, there have been several international genetically engineered bispecific antibody drugs that have been entered the clinical trial stage with a better application prospect.
5. Adoptive Immunotherapy of Tumors
As concerned in the adoptive immunotherapy of tumors, mainly comprising immunotherapy of LAK cells, TIL cells, activated T lymphocyte and CIK cells, autologous or allogeneic immunocompetent cells are delivered into the body of a patient after in vitro amplification to directly kill tumor cells, and regulate and enhance the immune function of the organism. However, the immunotherapy can be only used to remove a small number of scattered tumor cells and has a very limited effect on end-stage solid tumors, and is thus usually used as an adjuvant therapy to be combined with conventional methods, such as surgery, chemotherapy and radiotherapy. After a large number of tumor cells are cleared up by the conventional methods, residual tumor cells are removed by the immunotherapy, so that the comprehensive therapy effect on tumors can be improved. Wherein, as a new method for comprehensive therapy of tumors, the adoptive immunotherapy has been widely matched with conventional surgery, radiotherapy, chemotherapy and other cell and molecule therapies and holds great promise in therapy of multiple tumors. However, it should be a more ideal method that one end of the bispecific antibody can bind to a surface antigen CD3 of a cultured immune cell and is delivered into the body along with it, and the other end of the bispecific antibody can well bind to the surface antigen of the tumor cell; and therefore, the bispecific antibody can build a bridge between the tumor cell and the immune cell in the body, so that the immune cells are gathered around the tumor cells to further kill the tumor cells. By this method, the metastasis and diffusion of the tumor cells can be effectively solved, and the defects, such as ‘halfway, easy metastasis and large side effect’ in the three traditional therapy modes, namely surgery, radiotherapy and chemotherapy are overcome.